Loss of Pax3 causes reduction of melanocytes in the developing mouse cochlea

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of skin and retinal melanocytes, manifested as congenital hearing loss (~ 70%) and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. Cochlear melanocytes in the stria vascularis originated from Pax3-traced melanoblasts and Plp1-traced Schwann cell precursors, both of which derive from neural crest cells. Here, using a Pax3-Cre knock-in mouse that allows lineage tracing of Pax3-expressing cells and disruption of Pax3, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3+ derivatives contribute to S100+, Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all of which are significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in humans.

In this study, we examined Pax3-Cre knock-in mice 25 which allows fate-mapping of Pax3 expressing cells and ablation of the Pax3 gene, and found that complete loss of Pax3 prevents formation of melanocytes (intermediate cells) in the developing cochlea.We analyzed cochleae at E18.5 as Pax3 Cre/Cre homozygous mice were perinatally lethal 25 .At E18.5, Pax3 knockout cochleae showed 2 major phenotypes: 57% of animals showed smaller cochleae with normal organization of the organ of Corti, whereas 43% showed severely shortened cochlea in addition to gross organ dysgenesis such as exencephaly.Mice of both phenotypes lacked Pax3 protein expression.Furthermore, with fate-mapping and immunostaining for cochlear melanocytes, we uncovered that S100 + Pax3 Cre -EGFP + intermediate cells were diminished in the stria vascularis of E18.5 Pax3 knockout cochleae, with remaining intermediate cells continuing to differentiate into Dct + or Kir4.1 + melanocytes.Our results suggest that loss of Pax3 leads to reduction of cochlear melanocytes, which may contribute to congenital hearing loss in Waardenburg syndrome.

Results
Auditory function and cytoarchitecture of the Pax3 Cre/+ mice To determine the cell fate of Pax3 + derivatives in the cochlea, we used a lineage tracing approach (Pax3-Cre; CAG-CAT-EGFP or Rosa-mTmG) where Pax3-traced cells are labeled with EGFP.This approach has been used previously to fate-map derivatives of Pax3 + neuroepithelial cells including neural crest cells in various organs including the cochlea 5,6,25,26 .We first confirmed that Pax3 Cre -EGFP + cells were distributed primarily in the stria vascularis and modiolar regions, while occasionally EGFP + cells were detected in the organ of Corti and greater epithelial ridge (GER) region in the E18.5 and P1 Pax3 Cre/+ heterozygous cochleae (Fig. 1A,B).We also confirmed that Pax3 Cre/+ heterozygous embryos had normal cochlear development (Fig. 1A), consistent with prior reports 5,6 .Previously, Pax3 heterozygous mice (splotch mice, Sp) show normal hearing even though they have patchy pigmentation of skin hair like human Waardenburg syndrome patients 27 .We further assessed auditory responses of adult P56 Pax3 Cre/+ heterozygous mice with patchy pigmentation of skin hair and similarly found that they had auditory brain responses comparable to wildtype littermates at all frequencies tested (8, 16, 32 kHz) (Fig. 1C).
Like wildtype cochlea, the P43 Pax3 Cre/+ heterozygous cochlea displayed normal cytoarchitecture of the stria vascularis, organ of Corti and spiral ganglion (Fig. 1D,E).These data indicate that Pax3 Cre/+ heterozygous cochlea is grossly normal and may serve as a control for Pax3 Cre/Cre homozygous cochlea, and also as an excellent model to examine the Pax3 + derived cellular populations in the cochlea.

Pax3 knockout embryos show cochlear and vestibular defects in the late embryonic period
During normal development, the otic vesicle rapidly expands and gives rise to the cochlear duct and vestibular apparatus shortly after E10.5 [28][29][30] .Previously, Pax3 Cre/Cre homozygous embryos have been shown to display several developmental anomalies including loop tail and spina bifida with complete loss of Pax3 protein 25 .The degrees of anomaly are classified as mild or severe, with the latter showing exencephaly and shortened cochlea 5,6 .In this current study, Sox2 was expressed in the neuroepithelial layer of both Pax3 Cre/+ and Pax3 Cre/Cre brain (Supplementary Fig. S1A-C), and Pax3 Cre -EGFP + cells were found in the neural tube and roof plate of E11.5 Pax3 Cre/+ and Pax3 Cre/Cre mice with a mild phenotype (Supplementary Fig. S1A,B).Pax3 Cre -EGFP + cells were located in the lateral neural tube region of E11.5 Pax3 Cre/Cre mice with a severe phenotype including exencephaly (Supplementary Fig. S1C).At E11.5, Pax3 Cre/+ heterozygous mice expressed Pax3 in both Sox2 + and Pax3 Cre -EGFP + neural tube cells and roof plate cells (Supplementary Fig. S1A).In contrast, Pax3 expression was not detected in either Sox2 + neural tube cells or roof plate cells in E11.5 Pax3 Cre/Cre homozygous mice with a mild or severe phenotype, indicating that Pax3 was effectively ablated regardless of the severity of the phenotype (Supplementary Fig. S1B,C).Pax3 Cre/Cre homozygous embryos also die by P0 25 .Thus, we characterized the morphology of embryos shortly before birth at E18.5 (27 wildtype, 37 Pax3 Cre/+ heterozygous, 14 Pax3 Cre/Cre homozygous embryos from 7 litters) (Supplementary Fig. S2A-D, Table S1).We found that 8 Pax3 Cre/Cre homozygous embryos displayed mild anomalies (mostly loop tail and spina bifida) (Supplementary Fig. S2C, Table S1), and 6 were severe (visibly smaller, displayed exencephaly, loop tail, and spina bifida) (Supplementary Fig. S2D, Table S1).
To further examine the morphology of the inner ear of Pax3 Cre/Cre homozygous mice, we performed paint-fill of E15.5 cochleae, using wildtype as controls.The inner ear of E15.5 Pax3 Cre/Cre homozygous mice with a mild phenotype was smaller but exhibited similar morphology to wildtype control (Fig. 2A,B).However, Pax3 Cre/Cre homozygous embryo with a severe phenotype showed several malformations of both vestibular and cochlear organs, including underdeveloped semicircular canals, vestigial endolymphatic duct, and foreshortened cochlear duct (Fig. 2C).Similarly, at E18.5, Pax3 Cre/Cre homozygous inner ear with severe phenotype was noticeably smaller than wildtype, the Pax3 Cre/+ heterozygous inner ear and Pax3 Cre/Cre homozygous inner ear with mild phenotype (Fig. 2D-G).These results indicate that development of the inner ear is grossly normal in Pax3 Cre/Cre homozygous embryo with mild phenotype but is dramatically perturbed in Pax3 Cre/Cre homozygous embryo with the more generalized severe phenotype.

Some Pax3 + derivatives distribute as the intermediate cells in the stria vascularis of Pax3 knockout cochleae
Lineage tracing experiments using Wnt1-Cre, Plp1-Cre and Pax3-Cre mice demonstrate that neural crest cells migrate and develop as glial cells including Schwann cells and satellite cells in the spiral ganglion and cochlear melanocytes (intermediate cells) in the stria vascularis 5,6,8 .
The mature stria vascularis has three cell types: marginal cells, basal cells and intermediate cells, the latter of which are melanocytes.By contrast, the embryonic stria vascularis is composed of only marginal cells and intermediate cells 31 .Previously, Pax3 Cre/Cre homozygous cochlea has been shown to display a complete loss of Pax3 + derivatives and Dct + cochlear melanocytes in the stria vascularis at E15.5 6 .As melanocytes originate from both melanoblasts and Schwann cell precursors 8 , we hypothesize that Pax3 deficiency leads to a partial, and not complete, loss of cochlear melanocytes in the late embryonic period.First, we analyzed the distribution of the Pax3 + derivatives in the stria vascularis of the E18.5 Pax3 Cre/Cre homozygous cochlea.Kcnq1 is a marker for the marginal cells, and S100 marks both the marginal cells and intermediate cells in the stria vascularis 31,32 .In the www.nature.com/scientificreports/Pax3 Cre/+ heterozygous cochlea, many S100 + Pax3 Cre -EGFP + intermediate cells were detected next to Kcnq1 + S100 + marginal cells in the stria vascularis in all cochlear turns (Fig. 3A).By contrast, in the E18.5 Pax3 Cre/Cre homozygous cochlea with a mild and severe phenotype, rare or no S100 + Pax3 Cre -EGFP + intermediate cells were detected in the stria vascularis of the apical (Fig. 3Bʹʹ, Supplementary Fig. S4Aʹ), middle (Fig. 3Bʹ, Bʹʹʹʹ, Supplementary Fig. S4Aʹʹ) and basal turns (Fig. 3Bʹʹʹ, Supplementary Fig. S4Aʹʹʹ).Next, we quantified intermediate cells and compared the Pax3 Cre/Cre homozygous (mild phenotype) cochlea with that of the Pax3 Cre/+ heterozygous or wildtype cochlea at E18.5.All of them displayed four cochlear regions (apex, mid-apex, mid-base and base) in cross-section (Figs.1A, 3A,B, 4A,B, 5A,B, Supplementary Fig. S3A).In each cochlear turn, there were noticeably fewer S100 + Pax3 Cre -EGFP + intermediate cells in the stria vascularis of Pax3 Cre/Cre homozygous embryos with mild phenotype than Pax3 Cre/+ heterozygous embryos.The reduction is most dramatic in the apical turn relative to the base (Fig. 3C).Collectively, these data suggest that loss of Pax3 prevents normal development of intermediate cells in the cochlea, with the apical and middle cochlear turns more severely affected than the base.

Characterizing melanocytes in the stria vascularis in the Pax3 knockout embryos
To further characterize whether cochlear melanocytes were perturbed in Pax3 Cre/Cre homozygous mouse, we performed in situ hybridization for markers of melanocytes.Dct, a classical marker of melanocytes, was detected in all three turns in the E18.5 wildtype cochlea (Fig. 4A).In the Pax3 Cre/Cre homozygous cochlea (mild phenotype), we discovered markedly fewer Dct + melanocytes in all turns, with the greatest reduction observed in the apical turn (Fig. 4B,C).www.nature.com/scientificreports/Moreover, we examined expression of the inwardly rectifying potassium channel Kir4.1, whose expression in the stria vascularis is crucial for development of the endocochlear potential after P7 19,20,33 .Because Pax3 Cre/Cre homozygous mice are lethal perinatally, we investigated Kir4.1 mRNA expression in the E18.5 cochleae 25 .Kir4.1 mRNA was detected in the stria vascularis of both control and the Pax3 Cre/Cre homozygous mild phenotype cochlea.Kir4.1 mRNA was also detected in the organ of Corti and spiral ganglion (Fig. 5A,B).In the Pax3 Cre/Cre homozygous embryos with mild phenotype, there were significantly fewer Kir4.1 + cells in the stria vascularis in the mid-basal and basal turns than those in Pax3 Cre/+ heterozygous embryos, although the apical and mid-apical turns showed no significant reduction in the number of Kir4.1 + cells between those groups (Fig. 5C).Together, these data reveal that Pax3 deficiency perturbs development of cochlear melanocytes.

Discussion
Waardenburg syndrome is characterized by hearing loss and developmental abnormalities of melanocytes 12,34 .Genetic studies suggest that Waardenburg syndrome is caused by mutations of PAX3 and other genes such as MITF, SOX10, EDN3, EDNRB and SNAI2 1,9,13,[35][36][37] .Approximately 70% of Waardenburg syndrome patients suffer from sensorineural hearing loss through life and Pax3 is the most common causative mutation for Waardenburg syndrome (type 1 and 3) 18 .Here, we used a mouse model of Pax3 deficiency and found that loss of Pax3 causes a reduction of melanocytes in the developing cochlea, possibly stemming from a disruption to the distribution of neuroepithelial cells including neural crest cells.We showed that Pax3 Cre/+ heterozygous mice had normal cochlear development and no hearing loss, while Pax3 Cre/Cre homozygous mice showed fewer cochlear melanocytes (intermediate cells) which are required for normal hearing.Although the Pax3 knockout mice do not fully phenocopy Waardenburg syndrome in human, our and others' results 6,23 suggest that disruption of cochlear melanocytes as a result of Pax3 deficiency may contribute to their hearing loss.As a case in point, homozygous PAX3 mutations have been reported in type 3 Waardenburg syndrome patients [15][16][17] .Sensory epithelial cells in the inner ear are mostly derived from the otic vesicle 5,30 .Neuroepithelial cells including neural crest cells have also been proposed to contribute to the otic vesicle and later the sensory www.nature.com/scientificreports/epithelium 5 .Neural crest cells detached from the neural tube ectoderm migrate to the otic vesicle and differentiate into various cell types such as melanocytes, Schwann cells and satellite cells in the developing cochlea 38 .Humans carrying PAX3 mutations have abnormal development of melanocytes, manifested as heterochromia 1 .They also present with profound hearing loss despite grossly radiographically normal inner ear structures 24 .Previously, Pax3 mutants including Sp, Sp 2H and Pax3-Cre mice have been analyzed for cochlear development and those heterozygous mice are identified by the presence of patchy pigmentation of skin hair, which is one of the major phenotypes of type 1 and 3 Waardenburg syndrome patients 5,6,9,10,12,23,27 .Although type 1 and 3 Waardenburg syndrome patients with Pax3 heterozygous mutation typically exhibit severe-to-profound hearing loss, Sp heterozygous mice display normal hearing as do Pax3-Cre heterozygous mice in this study (Fig. 1C) 18,27 .Thus, the phenotype of heterozygous Pax3-Cre mice is less severe than in Pax3-heterozygous humans.In addition, we show that loss of Pax3 causes shortened cochlea and malformed vestibular apparatus using Pax3 Cre/Cre homozygous mice which are inserted with the Cre recombinase cDNA followed by a stop codon and a polyA signal in Pax3 exon 1, while having cochlear structures such as the stria vascularis 25 .Our study stands in contrast to the results of previous work using Sp 2H /Sp 2H homozygous embryo, in which 32 nucleotides deletion of Pax3 exon 5 by irradiation causes a truncated protein of its C-terminal half and prevents the formation of stria vascularis in the late embryonic cochlea 23,39,40 .This difference may be because the Sp 2H /Sp 2H homozygous mice still expressed some Pax3 protein whereas Pax3 Cre/Cre homozygous mice displayed no detectable Pax3 protein.Furthermore, www.nature.com/scientificreports/Pax3 Cre/Cre homozygous embryos in our study represent two distinct severities of phenotype.These diversities in phenotype may also be reflected in the variable degrees of hearing loss in type 1 and 3 Waardenburg syndrome caused by various PAX3 gene mutations [41][42][43] .
Neural crest cells with pluri-potent potential differentiate into the various cell types including melanocytes and play important roles in the development of various organs 2,25 .Melanocytes originating from neural crest cells migrate into specific locations within the skin and hair follicles, and to other sites including stria vascularis in the cochlea 44 .Cochlear melanocytes are known as intermediate cells which generate high concentration of potassium ions in the cochlear endolymph 20,45 .Endocochlear potential in-turn drives depolarization of hair cells and is required for hearing function 46 .One may hypothesize that the hearing loss associated with Waardenburg syndrome is a result of a disruption of the endocochlear potential arising from the developmental disorder of cochlear melanocytes, among other factors 6,27 .In this study, we demonstrated that Pax3 is necessary for the development of a full complement of cochlear melanocytes, with Pax3 deficiency leading to a reduction of cochlear melanocytes still expressing S100, Dct and Kir4.1 in the stria vascularis.Thus, our study pointed out that a small number of cochlear melanocytes can still develop despite Pax3 deficiency, suggesting the presence of alternative regulators of differentiation of Pax3 + derivatives.During development, Schwann cell precursors migrate into the stria vascularis starting at around E15.5 to give rise to cochlear melanocytes 8 .Interestingly, a previous report using Pax3-Cre knock-in mice 25 found no Pax3 + derivatives or Dct + melanocytes in the stria vascularis at E15.5, although the distribution of Pax3 + derivatives remained normal in the glial cell region 6 .With the same mouse line as the above report, we observed Pax3 + derivatives in both the glial cell region and stria vascularis, and Dct + melanocytes in the stria vascularis in the late embryonic period (E18.5).These divergent findings may be indicative of a delayed migration of Pax3 + derived cells from the glial region to other domains within the cochlea.
Finally, our data exhibited a degree of variance in the number of cochlear melanocytes in the stria vascularis caused by loss of Pax3.Whether this variance is indicative of the variable degree of hearing loss in Waardenburg syndrome remains unclear, as neither Pax3 heterozygous or homozygous mice phenocopy Waardenburg syndrome patients [41][42][43] .As such, a mouse model (e.g.Pax3 hypomorph) that more accurately models human Waardenburg syndrome is needed.The developing melanocytes in the human cochlea are considered as the prime target cells of gene therapy for Waardenburg syndrome 37 .Our results indicate that neuroepithelial cells with loss of Pax3 can differentiate as melanocytes if they properly migrate into the stria vascularis.In conclusion, our data would guide future studies to develop hearing therapies for Waardenburg syndrome.

Mice
The following mouse strains were used: Pax3 Cre/+ (Stock #005549, Jackson Laboratory) 25 , CAG CAT-EGFP/+ (gift from J. Miyazaki, Osaka Univ.) 47 , R26R mTmG mice (stock #007576, Jackson Laboratory) 48 .Mouse embryos of both genders were used.Institutional Animal Care and Use Committee of The Jikei University School of Medicine (protocol number: 21-025, 2020-060) and Stanford University School of Medicine (protocol number: 18606) approved all procedures.All experimental procedures were performed in accordance with relevant guidelines and regulations.This study is reported in accordance with ARRIVE guidelines, https:// arriv eguid elines.org.

Genotyping
Mouse genomic DNA was isolated from collected tail tips by adding 180 μl of 50 mM NaOH and incubating at 98 °C for 10 min, followed by the addition of 20 μl of 1 M Tris-HCl.PCR was performed to genotype transgenic mice with three specific primers which sequences were described in a previous paper 25 .

Auditory physiology measurements
Auditory brainstem responses were recorded as described in a previous paper 49 .Briefly, P56 mice were anesthetized with a ketamine/xylazine mixture (100 mg/kg ketamine and 10 mg/kg xylazine, IP) and placed on a heating pad at 37 °C.Auditory brain responses were measured with a needle electrode which was located inferior to the tympanic bulla, referenced to an electrode on the vertex of the head, and a ground electrode was inserted at the hind limb.Tone burst stimuli were delivered with frequencies ranging from 8 to 32 kHz (8.0, 16.0, 32.0 kHz) up to 90 dB sound pressure level (SPL) in 5 dB steps.At each frequency and SPL, 512 trials were tested and averaged.Two-way analysis of variance (ANOVA) with Sidak's multiple comparisons test was used for comparison of ABR thresholds.

Immunohistochemistry
Methods were modified as previously reported 50,51 .Briefly, E11.5 and E18.5 heads were harvested and fixed in 4% PFA overnight at 4 °C and then embedded in Tissue Tek OCT compound (Sakura, Tokyo, Japan) and frozen.Sections (10 μm thickness) were prepared using a cryostat CM3050S (Leica).P1 whole mount cochleae were dissected and fixed in 4% PFA for 1 h at room temperature (RT).Tissues were permeabilized with 0.5% TritonX-100 in PBS for 1 h at RT, and then blocked with 10% goat or donkey serum, 0.1% TritonX-100 and 1% bovine serum

Figure 4 .
Figure 4. Distribution of melanocytes in the Pax3 knockout cochleae at late embryonic day.(A-Aʹʹʹʹ) In the E18.5 Pax3 Cre/+ control cochlea, Dct + melanocytes were detected on a straight line along with the cochlear duct in the stria vascularis of all cochlear turns.(B-Bʹʹʹʹ) In the stria vascularis of the E18.5 Pax3 Cre/Cre mild phenotype cochlea, no Dct + melanocytes were found in the apical or mid-apical turn although a few Dct + melanocytes were detected in the mid-basal or basal turn.(C) Dct + melanocytes in stria vascularis were significantly fewer in all turns of the E18.5 Pax3 Cre/Cre mild phenotype homozygous cochleae than E18.5 wildtype cochleae.The basal turn had significantly more Dct + melanocytes than the apical and mid-apical turns in the E18.5 Pax3 Cre/Cre mild phenotype homozygous embryos.StV stria vascularis; data represent mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA with Tukey's multiple comparisons test).n = 6-7.